The present invention relates to a method for isolating cells and, more particularly, to a method for isolating "desired" cells from a population of cells, in which population the desired cells are admixed together with "undesired" cells.
In the context of the present invention, the "desired" cells are those living cells producing and releasing a particular product (e.g., antibody) or those living cells producing a particular molecule which, as opposed to being released from the cell, becomes affixed to the cell surface. For each respective case, "undesired" cells simply are those cells not possessing the noted characteristics of the desired cells.
In the various fields of biotechnology such as biochemistry, immunology, biogenetics, and the like, the art worker often is confronted with a cell population containing a large number of cells from which population it is desired to isolate those cells having certain characteristics, particularly the production and release of a desired product or the production of a particular molecule which becomes affixed to the cell surface. Typically, the ratio of these desired cells to the other undesired cells in the population is very small.
By way of illustration, the production of antibodies to a particular antigen typically relies upon the production of antibody by a mammal in response to an administered antigen or in response to a diseased state. Thus, cells removed from an animal or man which has received administered antigen, or which is diseased in such a way as to cause cells in the animal or man to have antigen on their surface, consist of a number of the desired cells, producing antibody, in admixture with many "undesired" cells which are not producing antibody. The isolation from this cell population of the desired, antibody-producing cells is laborious and difficult.
Similar difficulties are encountered in the production of monoclonal antibodies according to recently developed techniques such as hybridization. In hybridization, a collection of cells is harvested from an animal or man which has received administered antigen or which is diseased in a manner which causes cells to have antigen on their surface. The mixed cell collection so obtained contains desired antibody-producing cells in admixture with many cells not producing antibody. The cell population is mixed together with tumor cells such as myeloma cells, in the presence of a fusing agent such as polyethylene glycol, to bring about attachment between myeloma cells and the cells of the mixed cell population, from which attachments hybrid cells can form. Hybrid cells may be isolated from the population in a conventional manner; however, the isolation of desired hybrids, i.e., those hybrids formed between a myeloma cell and an antibody-producing cell and which, in fact, are producing and releasing antibody, is difficult and laborious.
Other situations exist in which the desired cell is one producing and releasing a product other than antibody, for example, an antigen or where the desired cell produces a molecule or product which becomes affixed to the cell surface rather than being released from the cell. In each instance, the isolation of these desired cells from other undesired cells with which they are mixed is difficult.
These difficulties in isolating desired cells from a population containing both these desired cells and undesired cells have spawned a few isolation techniques now practiced in the art. In one method, for isolating cells producing and releasing a desired product, individual cells in the cell population are removed and successively cloned so as to obtain a population of these cells of a size suitable for testing. The fluid in which the clone population is growing may then be tested for the presence of the desired product, a positive response indicating that the cells of the clone population are those making and releasing the desired product. As is apparent, however, this so-called "manual" method is inordinately time-consuming, particularly for typical cell populations containing many millions of cells.
In another method (the "Plaque assay"), suitable for use where the desired cell is one making and releasing an antibody, the population of desired and undesired cells is placed in a gel together with red blood cells having on their surface the appropriate antigen for the antibody released from the desired cells. The antibody released from the desired cells diffuses through the gel and attaches to the antigen on the surface of the red blood cells. The antigen-antibody complex now on the surface of the red blood cell makes it possible to lyse the red blood cell by addition of complement to the entire cell population. The lysed areas in the population can be seen (generally through microscopic examination) as clear areas, and the desired antibody-producing cells will be found in the vicinity (generally, the center) of the clear areas. Apart from being limited to the isolation of only one type of desired cells, this method also suffers from distinct limitations with respect to the number of cells which may be microscopically viewed at any given time and the difficulty of physically removing the desired cells from the indicated areas of the living cell population.
In yet another method, employed for isolating cells by virtue of a characteristic molecule produced by the cell and affixed to its surface, a ligand which will bind to the characteristic molecule is added to the cell population. The added ligand has fluorescein covalently attached thereto and, hence, the cells to which the ligand has attached (i.e., the desired cells) can be located in areas of the population found to be fluorescent. Suitable, automatic liquid-phase sorters exist which are capable of sorting areas in the population on the basis of the amount or quality of fluorescence. However, the selectivity of these sorters, owing to ever-present background "noise", is such that they are not capable of effective sorting where the desired cells constitute less than about 3% of the number of cells in the population being subjected to sorting. Moreover, these sorters are capable of sorting only up to about 1000 cells per second and, hence, an inordinate amount of time is required to completely scan and sort typical populations containing a large number of cells.
Another method known for isolating cells by virtue of a characteristic molecule which is produced by the cell and becomes affixed to its surface is to flow the cell population over a surface containing a suitable particle or substrate containing a ligand having an affinity for the molecule on the surface of the desired cell. Theoretically, the desired cells will thus be picked out from the flowing cell population by reason of their attachment to the ligand-carrying surface. However, this method of isolation is extremely difficult to perform satisfactorily in practice. On the one hand, if the binding affinity for the ligand to the molecule on the surface of the desired cells is not highly specific or strong, a number of undesired cells also will be captured by the ligand. On the other hand, a high binding affinity between ligand and molecule results in a pinching off of cells in the flowing population and an unwanted retention of desired cells.